Project Description: Sleep and sleep homeostasis are regulated by a network of sleep regulatory substances. A role for tumor necrosis factor ? (TNF) in sleep regulation is well characterized. However, TNF-TNF receptor interactions are complex; the mechanism involved in TNF-regulated sleep is unknown. Several electrophysiological parameters characterize sleep in animals; they include action potential burstiness (BI ? burstiness index), synchronization (SYN) of slow waves (SW) (0.5-3.5 Hz), SW power (V2) and evoked response potential (ERP) amplitudes. We use homologous measures to characterize sleep-like states in neuronal/glial co- cultures. Over the course of neuronal/glial culture development these parameters emerge as the networks mature. If cultured networks are stimulated electrically, these parameters decrease suggesting a more wake- like state. In contrast, if cultures are treated with TNF, these parameters increase indicating a deeper sleep- like state. Cultures also exhibit sleep homeostasis; after electrical stimulation a rebound increase in BI, SYN and SW power occurs. The effects of TNF on ERPs are stunning; TNF greatly enhances ERP amplitude and synchronization. We will use our in vitro sleep model to determine how TNF interacts with its receptors to affect culture state. In Aim 1, four experiments are proposed to determine which of the three known methods of TNF-TNF receptor interactions is responsible for the TNF-sleep actions. The TNF-TNF receptor interactions are: a) soluble TNF (17kD) acting as ligand for one of its receptors; b) trans-membrane TNF (26kD) directly binding to a TNF receptor on an adjacent cell to initiate responses (direct cell-to-cell signaling); and c) a soluble TNF receptor binding to trans-membrane TNF to initiate responses. In Aim 2 we determine which TNF receptor is involved. The in vitro system, due to its simplicity and our ability to control the intensity of the emergent state properties, offers a novel experimental platform to determine the mechanisms of action of TNF- sleep regulation and of emergent network properties. We use the R21 mechanism because the approach used, in vitro glial/neuronal cultures, is exploratory and in development and the experiments involve some risk yet have revolutionary potential (e.g. local sleep-like states being a consequence of direct cell-to-cell communication). As such they could transform basic sleep research and approaches to sleep clinical issues by providing a new, bottom-up approach to network emergent properties that have a role in practical sleep medicine problems, epilepsy, traumatic brain injury and other CNS disorders impacted by sleep.